Copyright 1998 by the Genetics Society of America Paramutation of the r1 Locus of Maize Is Associated With Increased Cytosine Methylation Elsbeth L. Walker Biology Department, University of Massachusetts, Amherst, Massachusetts 01003 Manuscript received September 25, 1997 Accepted for publication December 8, 1997 ABSTRACT In paramutation two alleles of a gene interact so that one of the alleles is epigenetically silenced. The silenced state is then genetically transmissible for many generations. The large (220 kbp) multigenic complex R-r is paramutable: its level of expression is changed during paramutation. R-r was found to exhibit increases in its level of cytosine methylation (C-methylation) following paramutation. These C-methylation changes are localized to the 59 portions of the two genes in the complex that are most sensitive to paramutation. These methylation changes ¯ank a small region called s that is thought to have been derived from a transposon named doppia. A mutant derivative of R-r that has a deletion of the s region fails to become methylated under conditions in which R-r is heavily methylated. This suggests that the presence of s sequences at the locus is required for the methylation changes that are observed following paramutation. ARAMUTATION is aregularly occurring, directed, pigmentation in which not all aleurone cells are colored. Pand heritable alteration of gene expression resulting The paramutant R-r complex is referred to as R-r9,to from the interaction of two alleles. One allele is referred distinguish it from nonparamutant R-r. Interestingly, to as paramutable: its expression changes following par- the effect on aleurone pigmentation is only observed if amutation. The other allele is paramutagenic: it causes R-r9 is transmitted through male gametes (pollen). the change in the paramutable allele. When the paramu- When R-r9 is transmitted through the female, the nor- table/paramutagenic heterozygote is crossed to allow mal dark uniform pigmentation pattern is observed. segregation of the two alleles, virtually 100% of the This dependence on the mode of transmission is not paramutable alleles transmitted display a decrease in due to a simple dosage effect; two paternally transmitted expression. This decreased expression level (the para- copies of R-r9 still confer a paramutant phenotype in mutant phenotype) persists through many generations. aleurone (Kermicle 1970). Nonparamutant R-r also ex- Paramutation was ®rst described in 1956 (Brink 1956), hibits mild silencing on male transmission. R-r9 is char- but a mechanism to explain how one allele can heritably acterized as being metastable; under certain conditions, affect the expression of another has remained obscure. it reverts toward the standard full color phenotype One of the best studied examples of paramutation is (Brink 1956; Brink 1958; Styles and Brink 1966; the r1 locus of Zea mays (Brink 1973; Kermicle 1996). Styles and Brink 1968). r1 genes encode helix-loop-helix proteins that are capa- The paramutable r1 complex, R-r, consists of four du- ble of directing transcription of the structural genes in plicated copies or partial copies of the r1 transcription the anthocyanin biosynthetic pathway (Goff et al. 1990; unit (Walker et al. 1995). These four copies, referred Ludwig et al. 1989). Thus, dominant R1 genes confer to as components, are designated P, q, S1, and S2. The red or purple anthocyanin pigmentation to the tissues S1 and S2 components are complete genes and are in which they are expressed. Paramutation at r1 is illus- responsible for pigmentation of the aleurone layer of trated in Figure 1. The crosses involve two r1 complexes, the seed. The q component is nonfunctional because it the paramutable R-r complex, and the paramutagenic lacks downstream coding sequences. The P component R-marbled (R-mb) complex. In the aleurone layer of the is the third complete r1 gene, and is active in several seed, the R-r complex normally confers full coloration. tissues in the plant including the coleoptile, the roots, The R-mb complex confers a marbled pattern of pigmen- and the anther walls. The three complete genes of the tation to aleurone. Following outcrossing of the hetero- complex are not equally subject to paramutation: the S zygote to a recessive colorless, r, tester strain, kernels genes (S1 and S2) show a large decrease in the amount receiving the R-r complex show a mottled pattern of of pigment they confer following paramutation, while the P gene shows a relatively small decrease following heterozygosity with a paramutagenic r1 complex (Brink Address for correspondence: Biology Department, Morrill Science Cen- Mikula Brown ter, University of Massachusetts, Amherst, MA 01003. and 1958; 1966). E-mail: [email protected] The S genes are arranged in an inverted head-to-head Genetics 148: 1973±1981 (April, 1998) 1974 E. L. Walker paramutation (Coe 1966; Hollick et al. 1995; Pat- terson et al. 1995). In petunia, a transgenic copy of the maize A1 gene can spontaneously become paramuta- genic (Meyer et al. 1993). Paramutation-like phenom- ena include a broad class of ªhomology-dependent gene silencingº events associated with meiotically stable re- ductions in gene expression (reviewed in Matzke et al. 1996; Meyer and Saedler 1996). Certain examples are particularly similar to paramutation since homologous gene copies interact in a way that heritably alters the expression of one of the copies, although the silencing and silenced genes are nonallelic. Examples of this type of interaction include the transgenic H locus in which an inactive transgene locus has the ability to silence partially homologous transgenes introduced into the same genome by genetic crossing (Matzke et al. 1994), and inactivation of endogenous genes that appears to occur among members of the phosphoribosylanthrani- Figure 1.ÐParamutation of r1. The series of crosses shown late synthase (PAI) gene family of the Wassilewskija involves three alleles of r1, the R-r complex (paramutable strain of Arabidopsis (Bender and Fink 1995). allele), the R-mb complex (paramutagenic allele), and a reces- Three features stand out as being held in common sive tester allele, r. R-r confers solid purple pigmentation to by several of these systems. One is that multiple copies the aleurone layer of the seeds; R-mb confers a marbled pattern of pigmentation to the aleurone; the r tester allele confers no of the genes are often present in the same genome color to aleurone. Paramutation is observed following test (reviewed in Flavell 1994; Matzke et al. 1996; Meyer crossing of the R-r/R-mb heterozygote. Half of the kernels on and Saedler 1996). A second feature is that inactivation the test cross ear carry the R-mb allele, which, when transmitted tends to be associated with increased cytosine methyla- through the female, confers a marbled pattern of pigmenta- tion (C-methylation) of both silenced and silencing loci tion in aleurone, but when transmitted through the male Bender Fink Eggleston Matzke confers extremely infrequent sectors rendering the aleurones ( and 1995; et al. 1995; mostly colorless. The R-r allele transmitted from the R-r/R- et al. 1994; Meyer et al. 1993; also reviewed in Matzke mb heterozygote is referred to as being ªparamutantº and is et al. 1996; Meyer and Saedler 1996). A ®nal feature designated as R-r9 to indicate this new epigenetic state. Male noted by some reviewers (Martienssen 1996a; Matzke transmission of R-r9 results in a pale mottled pattern of pig- mentation in the aleurone, while female transmission of R-r9 et al. 1996) is the possible involvement of transposable results in a normal solid color pattern of aleurone pigmenta- elements in some of these systems. Epigenetic inactiva- tion. tion of transposable elements is a well-known phenome- non (reviewed by Fedoroff 1996; Martienssen 1996b). Some paramutation systems, , and in maize, orientation and are separated by a 387-bp region called e.g. b1 pl1 do not share all of these features. Alleles of and s. The s region appears to be a rearranged remnant b1 pl1 Cone that are active in paramutation are simple, containing of a transposable element of the CACTA family ( Chandler et al. 1993; Upadhyaya et al. 1985) called doppia only a single gene copy ( et al. 1996). Further- (Walker et al. 1995). At its ends, s contains partial more, no C-methylation differences have been found Chandler copies of the terminal inverted repeats of the doppia during either b1 or pl1 paramutation ( et al. element. Internal to these inverted repeats are multiple 1996). Alleles of pl1 that are active in paramutation do Chandler copies of a subterminal repeated element of doppia, contain a doppia transposable element ( et al. Hollick and a second region consisting of sequences that have 1996; et al. 1995), but alleles of pl1 that are Coc- undergone extensive rearrangement and that may or inactive in paramutation also contain this element ( ciolone Cone may not have been derived from doppia. The s region and 1993). No transposable element has functions as the promoter for both the S1 and S2 genes been reported near or within the b1 alleles that are of R-r (Walker et al. 1995). doppia sequences that in- active in paramutation. In contrast, the r1 paramutation clude one terminal inverted repeat element and multi- system shares all three of these features. Both the para- ple subterminal repeats are also found distal to q mutable R-r complex (Robbins et al. 1991; Walker et (Walker et al. 1995). No doppia sequences are found al. 1995) and the paramutagenic R-mb (E. L. Walker, at the P component. unpublished results) and R-stippled (R-st)(Eggleston Recently, a variety of paramutation and paramutation- et al. 1995) complexes contain multiple r1 genes. Epige- like phenomena has been observed that can involve netic silencing of r1 genes has been correlated with either endogenous genes or transgenes.